The Toll-like receptors (TLRs) are important both because they mediate initial awareness of infection and because they elicit the most powerful inflammatory responses known. Both agonists and antagonists might be used for clinical purposes. Yet there is little understanding of how TLRs are triggered to yield qualitatively distinct signals, or what the rules for agonist and antagonist structure might be. We propose a collaboration between synthetic chemists and TLR biologists to identify and characterize new and inherently informative ligands for TLRs. The Boger lab has used solution-phase synthesis to produce a comprehensive library of peptide mimetic molecules, specifically designed to probe, mimic, and disrupt various types of protein-protein interactions. The Beutler lab has screened this library (95,000 compounds) for TLR agonists. Within an information-rich and comprehensive a-helix mimetic sub-library, several remarkably robust and structurally related hits have revealed a new class of TLR4 agonists. Studies of responses in germline mutant mice have shown that unlike LPS (the natural ligand for TLR4), these molecules signal to induce the NF-?B dependent cytokine TNF, but not type I IFNs. Structure-activity relationship (SAR) analyses have begun to reveal structural rules for agonist activity by these ligands, which conformationally resemble heptameric peptides with a-helical secondary structure. Continued screening of new libraries that are in progress will be performed to identify -turn, -sheet, and ?-turn peptidomimetic ligands with both agonist and antagonist properties. As they are discovered, these molecules will be subjected to SAR analysis, optimized for activity, and detailed studies of their effects on TLR signaling will be undertaken using TLR signaling mutants produced and maintained in the Beutler lab. The sites of interaction between these molecules and TLRs or associated proteins will be mapped using photoaffinity labeling methods. Where appropriate, the ligands will be co-crystallized with their cognate receptors for high resolution structural analysis. The properties of the molecules (pharmacokinetics, toxicity, immunoadjuvant effects, and ability to protect the host from model infections) will also be analyzed in vivo.